Publications by authors named "Daniel A Thomas"

34 Publications

Probing the conformational landscape and thermochemistry of DNA dinucleotide anions via helium nanodroplet infrared action spectroscopy.

Phys Chem Chem Phys 2020 Sep 14;22(33):18400-18413. Epub 2020 Aug 14.

Fritz-Haber-Institut der Max-Planck-Gesellschaft, Faradayweg 4-6, 14195 Berlin, Germany.

Isolation of biomolecules in vacuum facilitates characterization of the intramolecular interactions that determine three-dimensional structure, but experimental quantification of conformer thermochemistry remains challenging. Infrared spectroscopy of molecules trapped in helium nanodroplets is a promising methodology for the measurement of thermochemical parameters. When molecules are captured in a helium nanodroplet, the rate of cooling to an equilibrium temperature of ca. 0.4 K is generally faster than the rate of isomerization, resulting in "shock-freezing" that kinetically traps molecules in local conformational minima. This unique property enables the study of temperature-dependent conformational equilibria via infrared spectroscopy at 0.4 K, thereby avoiding the deleterious effects of spectral broadening at higher temperatures. Herein, we demonstrate the first application of this approach to ionic species by coupling electrospray ionization mass spectrometry (ESI-MS) with helium nanodroplet infrared action spectroscopy to probe the structure and thermochemistry of deprotonated DNA dinucleotides. Dinucleotide anions were generated by ESI, confined in an ion trap at temperatures between 90 and 350 K, and entrained in traversing helium nanodroplets. The infrared action spectra of the entrained ions show a strong dependence on pre-pickup ion temperature, consistent with the preservation of conformer population upon cooling to 0.4 K. Non-negative matrix factorization was utilized to identify component conformer infrared spectra and determine temperature-dependent conformer populations. Relative enthalpies and entropies of conformers were subsequently obtained from a van't Hoff analysis. IR spectra and conformer thermochemistry are compared to results from ion mobility spectrometry (IMS) and electronic structure methods. The implementation of ESI-MS as a source of dopant molecules expands the diversity of molecules accessible for thermochemical measurements, enabling the study of larger, non-volatile species.
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http://dx.doi.org/10.1039/d0cp02482aDOI Listing
September 2020

Design and Development of a Macrocyclic Series Targeting Phosphoinositide 3-Kinase δ.

ACS Med Chem Lett 2020 Jul 3;11(7):1386-1391. Epub 2020 Jun 3.

GSK Medicines Research Centre, Gunnels Wood Road, Stevenage SG1 2NY, United Kingdom.

A macrocyclization approach has been explored on a series of benzoxazine phosphoinositide 3-kinase δ inhibitors, resulting in compounds with improved potency, permeability, and clearance while maintaining good solubility. The thermodynamics of binding was explored via surface plasmon resonance, and the binding of lead macrocycle was found to be almost exclusively entropically driven compared with progenitor , which demonstrated both enthalpic and entropic contributions. The pharmacokinetics of macrocycle was also explored , where it showed reduced clearance when compared with the progenitor . This work adds to the growing body of evidence that macrocyclization could provide an alternative and complementary approach to the design of small-molecule inhibitors, with the potential to deliver differentiated properties.
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http://dx.doi.org/10.1021/acsmedchemlett.0c00061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7357223PMC
July 2020

Cryogenic Infrared Spectroscopy Reveals Structural Modularity in the Vibrational Fingerprints of Heparan Sulfate Diastereomers.

Anal Chem 2020 08 21;92(15):10228-10232. Epub 2020 Jul 21.

Department of Molecular Physics, Fritz Haber Institute of the Max Planck Society, Faradayweg 4-6, 14195 Berlin, Germany.

Heparan sulfate and heparin are highly acidic polysaccharides with a linear sequence, consisting of alternating glucosamine and hexuronic acid building blocks. The identity of hexuronic acid units shows a variability along their sequence, as d-glucuronic acid and its 5 epimer, l-iduronic acid, can both occur. The resulting backbone diversity represents a major challenge for an unambiguous structural assignment by mass spectrometry-based techniques. Here, we employ cryogenic infrared spectroscopy on mass-selected ions to overcome this challenge and distinguish isomeric heparan sulfate tetrasaccharides that differ only in the configuration of their hexuronic acid building blocks. High-resolution infrared spectra of a systematic set of synthetic heparan sulfate stereoisomers were recorded in the fingerprint region from 1000 to 1800 cm. The experiments reveal a characteristic combination of spectral features for each of the four diastereomers studied and imply structural modularity in the vibrational fingerprints. Strong spectrum-structure correlations were found and rationalized by state-of-the-art quantum chemical calculations. The findings demonstrate the potential of cryogenic infrared spectroscopy to extend the mass spectrometry-based toolkit for the sequencing of heparan sulfate and structurally related biomolecules.
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http://dx.doi.org/10.1021/acs.analchem.0c02048DOI Listing
August 2020

The Impact of Leaving Group Anomericity on the Structure of Glycosyl Cations of Protected Galactosides.

Chemphyschem 2020 09 30;21(17):1905-1907. Epub 2020 Jul 30.

Institute of Chemistry and Biochemistry, Freie Universität Berlin, Arnimallee 22, 14195, Berlin, Germany.

It has been reported that fragments produced by glycosidic bond breakage in mass spectrometry-based experiments can retain a memory of their anomeric configuration, which has major implications for glycan sequencing. Herein, we use cryogenic vibrational spectroscopy and ion mobility-mass spectrometry to study the structure of B-type fragments of protected galactosides. Cationic fragments were generated from glycosyl donors carrying trichloroacetimidate or thioethyl leaving groups of different anomeric configuration. The obtained infrared signatures indicate that the investigated fragments exhibit an identical structure, which suggests that there is no anomeric memory in B-type ions of fully protected monosaccharides.
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http://dx.doi.org/10.1002/cphc.202000473DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7540451PMC
September 2020

Remote Participation during Glycosylation Reactions of Galactose Building Blocks: Direct Evidence from Cryogenic Vibrational Spectroscopy.

Angew Chem Int Ed Engl 2020 04 2;59(15):6166-6171. Epub 2020 Mar 2.

Fritz-Haber-Institut der Max-Planck-Gesellschaft, Faradayweg 4-6, 14195, Berlin, Germany.

The stereoselective formation of 1,2-cis-glycosidic bonds is challenging. However, 1,2-cis-selectivity can be induced by remote participation of C4 or C6 ester groups. Reactions involving remote participation are believed to proceed via a key ionic intermediate, the glycosyl cation. Although mechanistic pathways were postulated many years ago, the structure of the reaction intermediates remained elusive owing to their short-lived nature. Herein, we unravel the structure of glycosyl cations involved in remote participation reactions via cryogenic vibrational spectroscopy and first principles theory. Acetyl groups at C4 ensure α-selective galactosylations by forming a covalent bond to the anomeric carbon in dioxolenium-type ions. Unexpectedly, also benzyl ether protecting groups can engage in remote participation and promote the stereoselective formation of 1,2-cis-glycosidic bonds.
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http://dx.doi.org/10.1002/anie.201916245DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7187407PMC
April 2020

Optimization of Orally Bioavailable PI3Kδ Inhibitors and Identification of Vps34 as a Key Selectivity Target.

J Med Chem 2020 01 8;63(2):638-655. Epub 2020 Jan 8.

Cellzome GmbH , GlaxoSmithKline , Meyerhofstrasse 1 , 69117 Heidelberg , Germany.

Optimization of a lead series of PI3Kδ inhibitors based on a dihydroisobenzofuran core led to the identification of potent, orally bioavailable compound . Selectivity profiling of compound showed similar potency for class III PI3K, Vps34, and PI3Kδ, and compound was not well-tolerated in a 7-day rat toxicity study. Structure-based design led to an improvement in selectivity for PI3Kδ over Vps34 and, a focus on oral phramacokinetics properties resulted in the discovery of compound , which showed improved toxicological outcomes at similar exposure levels to compound .
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http://dx.doi.org/10.1021/acs.jmedchem.9b01585DOI Listing
January 2020

IR action spectroscopy of glycosaminoglycan oligosaccharides.

Anal Bioanal Chem 2020 Jan 18;412(3):533-537. Epub 2019 Dec 18.

Department of Molecular Physics, Fritz Haber Institute of the Max Planck Society, Faradayweg 4-6, 14195, Berlin, Germany.

Glycosaminoglycans (GAGs) are a physio- and pharmacologically highly relevant class of complex saccharides, possessing a linear sequence and strongly acidic character. Their repetitive linear core makes them seem structurally simple at first glance, yet differences in sulfation and epimerization lead to an enormous structural diversity with only a few GAGs having been successfully characterized to date. Recent infrared action spectroscopic experiments on sulfated mono- and disaccharide ions show great promise. Here, we assess the potential of two types of gas-phase action spectroscopy approaches in the range from 1000 to 1800 cm for the structural analysis of complex GAG oligosaccharides. Synthetic tetra- and pentasaccharides were chosen as model compounds for this benchmark study. Utilizing infrared multiple photon dissociation action spectroscopy at room temperature, diagnostic bands are largely unresolved. In contrast, cryogenic infrared action spectroscopy of ions trapped in helium nanodroplets yields resolved infrared spectra with diagnostic features for monosaccharide composition and sulfation pattern. The analysis of GAGs could therefore significantly benefit from expanding the conventional MS-based toolkit with gas-phase cryogenic IR spectroscopy. Graphical abstract.
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http://dx.doi.org/10.1007/s00216-019-02327-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6992547PMC
January 2020

Resin and Magnetic Nanoparticle-Based Free Radical Probes for Glycan Capture, Isolation, and Structural Characterization.

Anal Chem 2019 12 25;91(24):15387-15396. Epub 2019 Nov 25.

Department of Chemistry and Biochemistry and Center for Quantitative Obesity Research , Montclair State University , Montclair , New Jersey 07043 , United States.

By combining the merits of solid supports and free radical activated glycan sequencing (FRAGS) reagents, we develop a multifunctional solid-supported free radical probe (SS-FRAGS) that enables glycan enrichment and characterization. SS-FRAGS comprises a solid support, free radical precursor, disulfide bond, pyridyl, and hydrazine moieties. Thio-activated resin and magnetic nanoparticles (MNPs) are chosen as the solid support to selectively capture free glycans via the hydrazine moiety, allowing for their enrichment and isolation. The disulfide bond acts as a temporary covalent linkage between the solid support and the captured glycan, allowing the release of glycans via the cleavage of the disulfide bond by dithiothreitol. The basic pyridyl functional group provides a site for the formation of a fixed charge, enabling detection by mass spectrometry and avoiding glycan rearrangement during collisional activation. The free radical precursor generates a nascent free radical upon collisional activation and thus simultaneously induces systematic and predictable fragmentation for glycan structure elucidation. A radical-driven glycan deconstruction diagram (R-DECON) is developed to visually summarize the MS results and thus allow for the assembly of the glycan skeleton, making the differentiation of isobaric glycan isomers unambiguous. For application to a real-world sample, we demonstrate the efficacy of the SS-FRAGS by analyzing glycan structures enzymatically cleaved from RNase-B.
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http://dx.doi.org/10.1021/acs.analchem.9b01303DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7537365PMC
December 2019

Characterization of a trans-trans Carbonic Acid-Fluoride Complex by Infrared Action Spectroscopy in Helium Nanodroplets.

J Am Chem Soc 2019 04 27;141(14):5815-5823. Epub 2019 Mar 27.

Fritz-Haber-Institut der Max-Planck-Gesellschaft , Faradayweg 4-6 , 14195 Berlin , Germany.

The high Lewis basicity and small ionic radius of fluoride promote the formation of strong ionic hydrogen bonds in the complexation of fluoride with protic molecules. Herein, we report that carbonic acid, a thermodynamically disfavored species that is challenging to investigate experimentally, forms a complex with fluoride in the gas phase. Intriguingly, this complex is highly stable and is observed in abundance upon nanoelectrospray ionization of an aqueous sodium fluoride solution in the presence of gas-phase carbon dioxide. We characterize the structure and properties of the carbonic acid-fluoride complex, F(HCO), and its deuterated isotopologue, F(DCO), by helium nanodroplet infrared action spectroscopy in the photon energy range of 390-2800 cm. The complex adopts a C symmetry structure with the carbonic acid in a planar trans-trans conformation and both OH groups forming ionic hydrogen bonds with the fluoride. Substantial vibrational anharmonic effects are observed in the infrared spectra, most notably a strong blue shift of the symmetric hydrogen stretching fundamental relative to predictions from the harmonic approximation or vibrational second-order perturbation theory. Ab initio thermostated ring-polymer molecular dynamics simulations indicate that this blue shift originates from strong coupling between the hydrogen stretching and bending vibrations, resulting in an effective weakening of the OH···F ionic hydrogen bonds.
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http://dx.doi.org/10.1021/jacs.8b13542DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6727381PMC
April 2019

The role of the mobile proton in fucose migration.

Anal Bioanal Chem 2019 Jul 2;411(19):4637-4645. Epub 2019 Mar 2.

Department of Molecular Physics, Fritz Haber Institute of the Max Planck Society, Faradayweg 4-6, 14195, Berlin, Germany.

Fucose migration reactions represent a substantial challenge in the analysis of fucosylated glycan structures by mass spectrometry. In addition to the well-established observation of transposed fucose residues in glycan-dissociation product ions, recent experiments show that the rearrangement can also occur in intact glycan ions. These results suggest a low-energy barrier for migration of the fucose residue and broaden the relevance of fucose migration to include other types of mass spectrometry experiments, including ion mobility-mass spectrometry and ion spectroscopy. In this work, we utilize cold-ion infrared spectroscopy to provide further insight into glycan scrambling in intact glycan ions. Our results show that the mobility of the proton is a prerequisite for the migration reaction. For the prototypical fucosylated glycans Lewis x and blood group antigen H-2, the formation of adduct ions or the addition of functional groups with variable proton affinity yields significant differences in the infrared spectra. These changes correlate well with the promotion or inhibition of fucose migration through the presence or absence of a mobile proton.
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http://dx.doi.org/10.1007/s00216-019-01657-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6611747PMC
July 2019

Fragment-Based Covalent Ligand Screening Enables Rapid Discovery of Inhibitors for the RBR E3 Ubiquitin Ligase HOIP.

J Am Chem Soc 2019 02 4;141(6):2703-2712. Epub 2019 Feb 4.

Molecular Structure of Cell Signalling Laboratory , The Francis Crick Institute , 1 Midland Road , London NW1 1AT , United Kingdom.

Modification of proteins with polyubiquitin chains is a key regulatory mechanism to control cellular behavior and alterations in the ubiquitin system are linked to many diseases. Linear (M1-linked) polyubiquitin chains play pivotal roles in several cellular signaling pathways mediating immune and inflammatory responses and apoptotic cell death. These chains are formed by the linear ubiquitin chain assembly complex (LUBAC), a multiprotein E3 ligase that consists of 3 subunits, HOIP, HOIL-1L, and SHARPIN. Herein, we describe the discovery of inhibitors targeting the active site cysteine of the catalytic subunit HOIP using fragment-based covalent ligand screening. We report the synthesis of a diverse library of electrophilic fragments and demonstrate an integrated use of protein LC-MS, biochemical ubiquitination assays, chemical synthesis, and protein crystallography to enable the first structure-based development of covalent inhibitors for an RBR E3 ligase. Furthermore, using cell-based assays and chemoproteomics, we demonstrate that these compounds effectively penetrate mammalian cells to label and inhibit HOIP and NF-κB activation, making them suitable hits for the development of selective probes to study LUBAC biology. Our results illustrate the power of fragment-based covalent ligand screening to discover lead compounds for challenging targets, which holds promise to be a general approach for the development of cell-permeable inhibitors of thioester-forming E3 ubiquitin ligases.
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http://dx.doi.org/10.1021/jacs.8b13193DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6383986PMC
February 2019

Publisher Correction: Unravelling the structure of glycosyl cations via cold-ion infrared spectroscopy.

Nat Commun 2018 11 8;9(1):4767. Epub 2018 Nov 8.

Department of Molecular Physics, Fritz Haber Institute of the Max Planck Society, Faradayweg 4-6, 14195, Berlin, Germany.

The original version of this Article contained an error in Fig. 1, in which an oxygen atom was missing from the 'Acetoxonium type' structure. This has been corrected in both the PDF and HTML versions of the Article.
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http://dx.doi.org/10.1038/s41467-018-07184-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6224614PMC
November 2018

Unravelling the structure of glycosyl cations via cold-ion infrared spectroscopy.

Nat Commun 2018 10 9;9(1):4174. Epub 2018 Oct 9.

Department of Molecular Physics, Fritz Haber Institute of the Max Planck Society, Faradayweg 4-6, 14195, Berlin, Germany.

Glycosyl cations are the key intermediates during the glycosylation reaction that covalently links building blocks during the synthetic assembly of carbohydrates. The exact structure of these ions remained elusive due to their transient and short-lived nature. Structural insights into the intermediate would improve our understanding of the reaction mechanism of glycosidic bond formation. Here, we report an in-depth structural analysis of glycosyl cations using a combination of cold-ion infrared spectroscopy and first-principles theory. Participating C2 protective groups form indeed a covalent bond with the anomeric carbon that leads to C1-bridged acetoxonium-type structures. The resulting bicyclic structure strongly distorts the ring, which leads to a unique conformation for each individual monosaccharide. This gain in mechanistic understanding fundamentally impacts glycosynthesis and will allow to tailor building blocks and reaction conditions in the future.
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http://dx.doi.org/10.1038/s41467-018-06764-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6177480PMC
October 2018

Ground-State Structure of the Proton-Bound Formate Dimer by Cold-Ion Infrared Action Spectroscopy.

Angew Chem Int Ed Engl 2018 Aug 12;57(33):10615-10619. Epub 2018 Jul 12.

Fritz-Haber-Institut der Max-Planck-Gesellschaft, Faradayweg 4-6, 14195, Berlin, Germany.

The proton-bound dicarboxylate motif, RCOO ⋅H ⋅ OOCR, is a prevalent chemical configuration found in many condensed-phase systems. The proton-bound formate dimer HCOO ⋅H ⋅ OOCH was studied utilizing cold-ion IR action spectroscopy in the range 400-1800 cm . The spectrum obtained at ca. 0.4 K of ions captured in He nanodroplets was compared to that measured at ca. 10 K by photodissociation of Ar-ion complexes. Similar band patterns are obtained by the two techniques that are consistent with calculations for a C symmetry structure with a proton shared equally between the two formate moieties. Isotopic substitution experiments point to the nominal parallel stretch of the bridging proton appearing as a sharp, dominant feature near 600 cm . Multidimensional anharmonic calculations reveal that the bridging proton motion is strongly coupled to the flanking -COO framework, an effect that is in line with the expected change in -C=O bond rehybridization upon protonation.
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http://dx.doi.org/10.1002/anie.201805436DOI Listing
August 2018

Fucose Migration in Intact Protonated Glycan Ions: A Universal Phenomenon in Mass Spectrometry.

Angew Chem Int Ed Engl 2018 06 25;57(25):7440-7443. Epub 2018 May 25.

Fritz-Haber-Institut der Max-Planck-Gesellschaft, Faradayweg 4-6, 14195, Berlin, Germany.

Fucose is an essential deoxysugar that is found in a wide range of biologically relevant glycans and glycoconjugates. A recurring problem in mass spectrometric analyses of fucosylated glycans is the intramolecular migration of fucose units, which can lead to erroneous sequence assignments. This migration reaction is typically assigned to activation during collision-induced dissociation (CID) in tandem mass spectrometry (MS). In this work, we utilized cold-ion spectroscopy and show for the first time that fucose migration is not limited to fragments obtained in tandem MS and can also be observed in intact glycan ions. This observation suggests a possible low-energy barrier for this transfer reaction and generalizes fucose migration to an issue that may universally occur in any type of mass spectrometry experiment.
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http://dx.doi.org/10.1002/anie.201801418DOI Listing
June 2018

Vibrational Spectroscopy of Fluoroformate, FCO, Trapped in Helium Nanodroplets.

J Phys Chem Lett 2018 May 23;9(9):2305-2310. Epub 2018 Apr 23.

Fritz-Haber-Institut der Max-Planck-Gesellschaft , Faradayweg 4-6 , 14195 Berlin , Germany.

Fluoroformate, also known as carbonofluoridate, is an intriguing molecule readily formed by the reductive derivatization of carbon dioxide. In spite of its well-known stability, a detailed structural characterization of the isolated anion has yet to be reported. Presented in this work is the vibrational spectrum of fluoroformate obtained by infrared action spectroscopy of ions trapped in helium nanodroplets, the first application of this technique to a molecular anion. The experimental method yields narrow spectral lines, providing experimental constraints on the structure that can be accurately reproduced using high-level ab initio methods. In addition, two notable Fermi resonances between a fundamental and combination band are observed. The electrostatic potential map of fluoroformate reveals substantial charge density on fluorine as well as on the oxygen atoms, suggesting multiple sites for interaction with hydrogen bond donors and electrophiles, which may in turn lead to intriguing solvation structures and reaction pathways.
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http://dx.doi.org/10.1021/acs.jpclett.8b00664DOI Listing
May 2018

Selectively Targeting the Kinome-Conserved Lysine of PI3Kδ as a General Approach to Covalent Kinase Inhibition.

J Am Chem Soc 2018 01 9;140(3):932-939. Epub 2018 Jan 9.

Medicines Research Centre, GlaxoSmithKline , Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, U.K.

Selective covalent inhibition of kinases by targeting poorly conserved cysteines has proven highly fruitful to date in the development of chemical probes and approved drugs. However, this approach is limited to ∼200 kinases possessing such a cysteine near the ATP-binding pocket. Herein, we report a novel approach to achieve selective, irreversible kinase inhibition, by targeting the conserved catalytic lysine residue. We have illustrated our approach by developing selective, covalent PI3Kδ inhibitors that exhibit nanomolar potency in cellular assays, and a duration of action >48 h in CD4+ T cells. Despite conservation of the lysine residue throughout the kinome, the lead compound shows high levels of selectivity over a selection of lipid and protein kinases in biochemical assays, as well as covalent binding to very few off-target proteins in live-cell proteomic studies. We anticipate this approach could offer a general strategy, as an alternative to targeting non-conserved cysteines, for the development of selective covalent kinase inhibitors.
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http://dx.doi.org/10.1021/jacs.7b08979DOI Listing
January 2018

From PIM1 to PI3Kδ via GSK3β: Target Hopping through the Kinome.

ACS Med Chem Lett 2017 Oct 7;8(10):1093-1098. Epub 2017 Sep 7.

Molecular Discovery Research, Biological Sciences, and Computational Chemistry, Platform Technology & Science, GlaxoSmithKline R&D, Gunnels Wood Road, Stevenage, SG1 2NY, U.K.

Selective inhibitors of phosphoinositide 3-kinase delta are of interest for the treatment of inflammatory diseases. Initial optimization of a 3-substituted indazole hit compound targeting the kinase PIM1 focused on improving selectivity over GSK3β through consideration of differences in the ATP binding pockets. Continued kinase cross-screening showed PI3Kδ activity in a series of 4,6-disubstituted indazole compounds, and subsequent structure-activity relationship exploration led to the discovery of an indole-containing lead compound as a potent PI3Kδ inhibitor with selectivity over the other PI3K isoforms.
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http://dx.doi.org/10.1021/acsmedchemlett.7b00296DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5642016PMC
October 2017

Glycan Fingerprinting via Cold-Ion Infrared Spectroscopy.

Angew Chem Int Ed Engl 2017 09 8;56(37):11248-11251. Epub 2017 Jun 8.

Fritz-Haber-Institut der Max-Planck-Gesellschaft, Faradayweg 4-6, 14195, Berlin, Germany.

The diversity of stereochemical isomers present in glycans and glycoconjugates poses a formidable challenge for comprehensive structural analysis. Typically, sophisticated mass spectrometry (MS)-based techniques are used in combination with chromatography or ion-mobility separation. However, coexisting structurally similar isomers often render an unambiguous identification impossible. Other powerful techniques such as gas-phase infrared (IR) spectroscopy have been limited to smaller glycans, since conformational flexibility and thermal activation during the measurement result in poor spectral resolution. This limitation can be overcome by using cold-ion spectroscopy. The vibrational fingerprints of cold oligosaccharide ions exhibit a wealth of well-resolved absorption features that are diagnostic for minute structural variations. The unprecedented resolution of cold-ion spectroscopy coupled with tandem MS may render this the key technology to unravel complex glycomes.
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http://dx.doi.org/10.1002/anie.201702896DOI Listing
September 2017

Real-Time Studies of Iron Oxalate-Mediated Oxidation of Glycolaldehyde as a Model for Photochemical Aging of Aqueous Tropospheric Aerosols.

Environ Sci Technol 2016 11 25;50(22):12241-12249. Epub 2016 Oct 25.

Arthur Amos Noyes Laboratory of Chemical Physics, California Institute of Technology , Pasadena, California 91125, United States.

The complexation of iron(III) with oxalic acid in aqueous solution yields a strongly absorbing chromophore that undergoes efficient photodissociation to give iron(II) and the carbon dioxide anion radical. Importantly, iron(III) oxalate complexes absorb near-UV radiation (λ > 350 nm), providing a potentially powerful source of oxidants in aqueous tropospheric chemistry. Although this photochemical system has been studied extensively, the mechanistic details associated with its role in the oxidation of dissolved organic matter within aqueous aerosol remain largely unknown. This study utilizes glycolaldehyde as a model organic species to examine the oxidation pathways and evolution of organic aerosol initiated by the photodissociation of aqueous iron(III) oxalate complexes. Hanging droplets (radius 1 mm) containing iron(III), oxalic acid, glycolaldehyde, and ammonium sulfate (pH ∼3) are exposed to irradiation at 365 nm and sampled at discrete time points utilizing field-induced droplet ionization mass spectrometry (FIDI-MS). Glycolaldehyde is found to undergo rapid oxidation to form glyoxal, glycolic acid, and glyoxylic acid, but the formation of high molecular weight oligomers is not observed. For comparison, particle-phase experiments conducted in a laboratory chamber explore the reactive uptake of gas-phase glycolaldehyde onto aqueous seed aerosol containing iron and oxalic acid. The presence of iron oxalate in seed aerosol is found to inhibit aerosol growth. These results suggest that photodissociation of iron(III) oxalate can lead to the formation of volatile oxidation products in tropospheric aqueous aerosols.
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http://dx.doi.org/10.1021/acs.est.6b03588DOI Listing
November 2016

Eradicating mass spectrometric glycan rearrangement by utilizing free radicals.

Chem Sci 2016 Aug 5;7(8):5390-5397. Epub 2016 May 5.

Arthur Amos Noyes Laboratory of Chemical Physics , California Institute of Technology , 1200 East California Blvd , Pasadena , CA 91125 , USA . Email:

Mass spectrometric glycan rearrangement is problematic because it provides misleading structural information. Here we report on a new reagent, a methylated free radical activated glycan sequencing reagent (Me-FRAGS), which combines a free radical precursor with a methylated pyridine moiety that can be coupled to the reducing terminus of glycans. The collisional activation of Me-FRAGS-derivatized glycans generates a nascent free radical that concurrently induces abundant glycosidic bond and cross-ring cleavage without the need for subsequent activation. The product ions resulting from glycan rearrangement, including internal residue loss and multiple external residue losses, are precluded. Glycan structures can be easily assembled and visualized using a radical driven glycan deconstruction diagram (R-DECON diagram). The presence and location of -acetylated saccharide units and branch sites can be identified from the characteristic dissociation patterns observed only at these locations. The mechanisms of dissociation are investigated and discussed. This Me-FRAGS based mass spectrometric approach creates a new blueprint for glycan structure analysis.
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http://dx.doi.org/10.1039/c6sc01371fDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6020757PMC
August 2016

Optimization of Novel Indazoles as Highly Potent and Selective Inhibitors of Phosphoinositide 3-Kinase δ for the Treatment of Respiratory Disease.

J Med Chem 2015 Sep 3;58(18):7381-99. Epub 2015 Sep 3.

Refractory Respiratory Inflammation DPU, and ‡Allergic Inflammation DPU, Respiratory Therapeutic Area, GlaxoSmithKline R&D , Gunnels Wood Road, Stevenage, SG1 2NY, U.K.

Optimization of lead compound 1, through extensive use of structure-based design and a focus on PI3Kδ potency, isoform selectivity, and inhaled PK properties, led to the discovery of clinical candidates 2 (GSK2269557) and 3 (GSK2292767) for the treatment of respiratory indications via inhalation. Compounds 2 and 3 are both highly selective for PI3Kδ over the closely related isoforms and are active in a disease relevant brown Norway rat acute OVA model of Th2-driven lung inflammation.
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http://dx.doi.org/10.1021/acs.jmedchem.5b00767DOI Listing
September 2015

Mechanisms and energetics of free radical initiated disulfide bond cleavage in model peptides and insulin by mass spectrometry.

Chem Sci 2015 Aug 20;6(8):4550-4560. Epub 2015 May 20.

Division of Chemistry and Chemical Engineering , California Institute of Technology , Pasadena , CA 91125 , USA . Email:

We investigate the mechanism of disulfide bond cleavage in gaseous peptide and protein ions initiated by a covalently-attached regiospecific acetyl radical using mass spectrometry (MS). Highly selective S-S bond cleavages with some minor C-S bond cleavages are observed by a single step of collisional activation. We show that even multiple disulfide bonds in intact bovine insulin are fragmented in the MS2 stage, releasing the A- and B-chains with a high yield, which has been challenging to achieve by other ion activation methods. Yet, regardless of the previous reaction mechanism studies, it has remained unclear why (1) disulfide bond cleavage is preferred to peptide backbone fragmentation, and why (2) the S-S bond that requires the higher activation energy conjectured in previously suggested mechanisms is more prone to be cleaved than the C-S bond by hydrogen-deficient radicals. To probe the mechanism of these processes, model peptides possessing deuterated β-carbon(s) at the disulfide bond are employed. It is suggested that the favored pathway of S-S bond cleavage is triggered by direct acetyl radical attack at sulfur with concomitant cleavage of the S-S bond (S2). The activation energy for this process is substantially lower by ∼9-10 kcal mol than those of peptide backbone cleavage processes determined by density functional quantum chemical calculations. Minor reaction pathways are initiated by hydrogen abstraction from the α-carbon or the β-carbon of a disulfide, followed by β-cleavages yielding C-S or S-S bond scissions. The current mechanistic findings should be generally applicable to other radical-driven disulfide bond cleavages with different radical species such as the benzyl and methyl pyridyl radicals.
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http://dx.doi.org/10.1039/c5sc01305dDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5666513PMC
August 2015

Mass spectrometric sampling of a liquid surface by nanoliter droplet generation from bursting bubbles and focused acoustic pulses: application to studies of interfacial chemistry.

Anal Chem 2015 Mar 4;87(6):3336-44. Epub 2015 Mar 4.

†Arthur Amos Noyes Laboratory of Chemical Physics, California Institute of Technology, Pasadena, California 91125, United States.

The complex chemistry occurring at the interface between liquid and vapor phases contributes significantly to the dynamics and evolution of numerous chemical systems of interest, ranging from damage to the human lung surfactant layer to the aging of atmospheric aerosols. This work presents two methodologies to eject droplets from a liquid water surface and analyze them via mass spectrometry. In bursting bubble ionization (BBI), droplet ejection is achieved via the formation of a jet following bubble rupture at the surface of a liquid to yield 250 μm diameter droplets (10 nL volume). In interfacial sampling by an acoustic transducer (ISAT), droplets are produced by focusing pulsed piezoelectric transducer-generated acoustic waves at the surface of a liquid, resulting in the ejection of droplets of 100 μm in diameter (500 pL volume). In both experimental methodologies, ejected droplets are aspirated into the inlet of the mass spectrometer, resulting in the facile formation of gas-phase ions. We demonstrate the ability of this technique to readily generate spectra of surface-active analytes, and we compare the spectra to those obtained by electrospray ionization. Charge measurements indicate that the ejected droplets are near-neutral (<0.1% of the Rayleigh limit), suggesting that gas-phase ion generation occurs in the heated transfer capillary of the instrument in a mechanism similar to thermospray or sonic spray ionization. Finally, we present the oxidation of oleic acid by ozone as an initial demonstration of the ability of ISAT-MS to monitor heterogeneous chemistry occurring at a planar water/air interface.
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http://dx.doi.org/10.1021/ac504494tDOI Listing
March 2015

Hydrogen bonding constrains free radical reaction dynamics at serine and threonine residues in peptides.

J Phys Chem A 2014 Sep 20;118(37):8380-92. Epub 2014 Mar 20.

Arthur Amos Noyes Laboratory of Chemical Physics, California Institute of Technology , Pasadena, California 91125, United States.

Free radical-initiated peptide sequencing (FRIPS) mass spectrometry derives advantage from the introduction of highly selective low-energy dissociation pathways in target peptides. An acetyl radical, formed at the peptide N-terminus via collisional activation and subsequent dissociation of a covalently attached radical precursor, abstracts a hydrogen atom from diverse sites on the peptide, yielding sequence information through backbone cleavage as well as side-chain loss. Unique free-radical-initiated dissociation pathways observed at serine and threonine residues lead to cleavage of the neighboring N-terminal Cα-C or N-Cα bond rather than the typical Cα-C bond cleavage observed with other amino acids. These reactions were investigated by FRIPS of model peptides of the form AARAAAXAA, where X is the amino acid of interest. In combination with density functional theory (DFT) calculations, the experiments indicate the strong influence of hydrogen bonding at serine or threonine on the observed free radical chemistry. Hydrogen bonding of the side-chain hydroxyl group with a backbone carbonyl oxygen aligns the singly occupied π orbital on the β-carbon and the N-Cα bond, leading to low-barrier β-cleavage of the N-Cα bond. Interaction with the N-terminal carbonyl favors a hydrogen-atom transfer process to yield stable c and z(•) ions, whereas C-terminal interaction leads to effective cleavage of the Cα-C bond through rapid loss of isocyanic acid. Dissociation of the Cα-C bond may also occur via water loss followed by β-cleavage from a nitrogen-centered radical. These competitive dissociation pathways from a single residue illustrate the sensitivity of gas-phase free radical chemistry to subtle factors such as hydrogen bonding that affect the potential energy surface for these low-barrier processes.
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http://dx.doi.org/10.1021/jp501367wDOI Listing
September 2014

Omental Torsion: An Unusual Cause of Right Iliac Fossa Pain and Role of Laparoscopic Management.

Gastroenterology Res 2013 Dec 15;6(6):237-239. Epub 2014 Jan 15.

Department of Surgery, Queen Mary Hospital, Sidcup, Kent DA14, UK.

Omental torsion is a rare cause of acute abdomen. It usually presents with acute onset right-sided abdominal pain. Adult male between 40 and 50 years of age and obesity are the most common risk factor amongst others. Clinical diagnosis is challenging and difficult to differentiate from more common clinical pathologies such as acute appendicitis and/or acute cholecystitis. Transabdominal imagings such as ultrasonography and/or computed tomography are useful showing typical whirl pattern. Advocated management is surgical excision of torted omentum. Herein, we report a case of primary omental torsion in an adult and a review of current literature. The diagnosis was incidental when patient was undertaken for laparoscopic appendectomy. Only the distal edge of right omentum was torted making a fatty mass of 4 × 3 cm lying on the ascending colon that could have been easily missed if open appendectomy was opted. This case not only highlights the importance of considering torted omentum in differential diagnosis of right-sided abdominal pains but also backs the changing practice to laparoscopic approach for management of right iliac fossa pain.
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http://dx.doi.org/10.4021/gr299eDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5051132PMC
December 2013

Biomimetic reagents for the selective free radical and acid-base chemistry of glycans: application to glycan structure determination by mass spectrometry.

J Am Chem Soc 2013 Jul 12;135(29):10684-92. Epub 2013 Jul 12.

Arthur Amos Noyes Laboratory of Chemical Physics, California Institute of Technology, Pasadena, California 91125, USA.

Nature excels at breaking down glycans into their components, typically via enzymatic acid-base catalysis to achieve selective cleavage of the glycosidic bond. Noting the importance of proton transfer in the active site of many of these enzymes, we describe a sequestered proton reagent for acid-catalyzed glycan sequencing (PRAGS) that derivatizes the reducing terminus of glycans with a pyridine moiety possessing moderate proton affinity. Gas-phase collisional activation of PRAGS-derivatized glycans predominately generates C1-O glycosidic bond cleavages retaining the charge on the reducing terminus. The resulting systematic PRAGS-directed deconstruction of the glycan can be analyzed to extract glycan composition and sequence. Glycans are also highly susceptible to dissociation by free radicals, mainly reactive oxygen species, which inspired our development of a free radical activated glycan sequencing (FRAGS) reagent, which combines a free radical precursor with a pyridine moiety that can be coupled to the reducing terminus of target glycans. Collisional activation of FRAGS-derivatized glycans generates a free radical that reacts to yield abundant cross-ring cleavages, glycosidic bond cleavages, and combinations of these types of cleavages with retention of charge at the reducing terminus. Branched sites are identified with the FRAGS reagent by the specific fragmentation patterns that are observed only at these locations. Mechanisms of dissociation as well as application of the reagents for both linear and highly branched glycan structure analysis are investigated and discussed. The approach developed here for glycan structure analysis offers unique advantages compared to earlier studies employing mass spectrometry for this purpose.
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http://dx.doi.org/10.1021/ja402810tDOI Listing
July 2013

Ion mobility-mass spectrometry with a radial opposed migration ion and aerosol classifier (ROMIAC).

Anal Chem 2013 Jul 19;85(13):6319-26. Epub 2013 Jun 19.

Division of Engineering and Applied Science, California Institute of Technology, Pasadena, California 91125, USA.

The first application of a novel differential mobility analyzer, the radial opposed migration ion and aerosol classifier (ROMIAC), is demonstrated. The ROMIAC uses antiparallel forces from an electric field and a cross-flow gas to both scan ion mobilities and continuously transmit target mobility ions with 100% duty cycle. In the ROMIAC, diffusive losses are minimized, and resolution of ions, with collisional cross-sections of 200-2000 Å(2), is achieved near the nondispersive resolution of ~20. Higher resolution is theoretically possible with greater cross-flow rates. The ROMIAC was coupled to a linear trap quadrupole mass spectrometer and used to classify electrosprayed C2-C12 tetra-alkyl ammonium ions, bradykinin, angiotensin I, angiotensin II, bovine ubiquitin, and two pairs of model peptide isomers. Instrument and mobility calibrations of the ROMIAC show that it exhibits linear responses to changes in electrode potential, making the ROMIAC suitable for mobility and cross-section measurements. The high resolution of the ROMIAC facilitates separation of isobaric isomeric peptides. Monitoring distinct dissociation pathways associated with peptide isomers fully resolves overlapping peaks in the ion mobility data. The ability of the ROMIAC to operate at atmospheric pressure and serve as a front-end analyzer to continuously transmit ions with a particular mobility facilitates extensive studies of target molecules using a variety of mass spectrometric methods.
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http://dx.doi.org/10.1021/ac400580uDOI Listing
July 2013

Discovery and biochemical characterization of Plasmodium thioredoxin reductase inhibitors from an antimalarial set.

Biochemistry 2012 Jun 1;51(23):4764-71. Epub 2012 Jun 1.

GlaxoSmithKline, Medicines Research Centre, Stevenage, Hertfordshire, UK.

Plasmodium falciparum is the most prevalent and deadly species of the human malaria parasites, and thioredoxin reductase (TrxR) is an enzyme involved in the redox response to oxidative stress. Essential for P. falciparum survival, the enzyme has been highlighted as a promising target for novel antimalarial drugs. Here we report the discovery and characterization of seven molecules from an antimalarial set of 13533 compounds through single-target TrxR biochemical screens. We have produced high-purity, full-length, recombinant native enzyme from four Plasmodium species, and thioredoxin substrates from P. falciparum and Rattus norvegicus. The enzymes were screened using a unique, high-throughput, in vitro native substrate assay, and we have observed selectivity between the Plasmodium species and the mammalian form of the enzyme. This has indicated differences in their biomolecular profiles and has provided valuable insights into the biochemical mechanisms of action of compounds with proven antimalarial activity.
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http://dx.doi.org/10.1021/bi3005076DOI Listing
June 2012

High-yield production and characterization of biologically active GST-tagged human topoisomerase IIα protein in insect cells for the development of a high-throughput assay.

Protein Expr Purif 2011 Apr 13;76(2):165-72. Epub 2010 Aug 13.

Biological Reagents & Assay Development, GlaxoSmithKline R&D, New Frontiers Science Park, Harlow, Essex, UK.

DNA topoisomerase type II enzymes are well-validated targets of anti-bacterial and anti-cancer compounds. In order to facilitate discovery of these types of inhibitors human topoisomerase II in vitro assays can play an important role to support drug discovery processes. Typically, human topoisomerase IIα proteins have been purified from human cell lines or as untagged proteins from yeast cells. This study reports a method for the rapid over-expression and purification of active GST-tagged human topoisomerase IIα using the baculovirus mediated insect cell expression system. Expression of the GST fused protein was observed in the nuclear fraction of insect cells. High yields (40 mg/L i.e. 8 mg/10(9) cells) at >80% purity of this target was achieved by purification using a GST HiTrap column followed by size exclusion chromatography. Functional activity of GST-tagged human topoisomerase IIα was demonstrated by ATP-dependent relaxation of supercoiled DNA in an agarose gel based assay. An 8-fold DNA-dependent increase in ATPase activity of this target compared to its intrinsic activity was also demonstrated in a high-throughput ATPase fluorescence based assay. Human topoisomerase IIα inhibitors etoposide, quercetin and suramin were tested in the fluorescence assay. IC(50) values obtained were in good agreement with published data. These inhibitors also demonstrated ≥ 30-fold potency over the anti-bacterial topoisomerase II inhibitor ciprofloxacin in the assay. Collectively these data validated the enzyme and the high-throughput fluorescence assay as tools for inhibitor identification and selectivity studies.
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http://dx.doi.org/10.1016/j.pep.2010.08.001DOI Listing
April 2011